This invention relates to bioluminescent proteins, in particular it relates to bioluminescent proteins which have been modified, for example by chemical means or by genetic engineering. Such modified bioluminescent proteins, hereinafter referred to as xe2x80x9crainbow proteinsxe2x80x9d, may be used in the detection and quantification of cells, microbes such as bacteria, viruses and protozoa, and substances of biological interest such as substrates, metabolites, intra- and extra- cellular signals, enzymes, antigens, antibodies and nucleic acids.
Bioluminescence is the oxidation of an organic molecule, the xe2x80x9cluciferinxe2x80x9d, by oxygen or one of its metabolites, to emit light. The reaction is catalysed by a protein, usually known as a xe2x80x9cluciferasexe2x80x9d, or a xe2x80x9cphotoproteinxe2x80x9d when the luciferin is so tightly or covalently bound to the luciferase that it does not diffuse off into the surrounding fluid.
O2+lucifern+luciferasexe2x86x92oxyluciferin+light (or O{overscore (2+L )} or H2O2) (or photoprotein) to three other substances may also be required to be present in order to generate light, or to alter its colour, and they are as follows:
(a) A cation such as H+, Ca2+, Mg2+, or a transition metal such as Cu+/Cu2+, Fe2+/Fe3+.
(b) A cofactor such as NADH, FMU, or ATP.
(c) A fluor as an energy transfer acceptor.
Five chemical families of luciferin have been identified so far (see FIG. 1 of the attached drawing):
(a) Aldehydes (found in bacteria, freshwater limpet Latia and earthworms).
(b) Imidazolopyrazines (found in Sarcomastigophora, Cnidaria, Ctenophora, some Arthropoda, some Mollusca, some Chordata).
(c) Benzothiazole (found in beetles such as fireflies and glowworms).
(d) Linear tetrapyrroles (found in dinoflagellates, euphasiid shrimp, some fish).
(e) Flavins (found in bacteria, fungi, polychaete worms and some molluscs).
Reactions involving these luciferins may result in the emission of violet, blue, blue-green, green, yellow or red light and occasionally UV or IR light and such emission may or may not be linearly or circularly polarised. Reference is directed to Chemiluminescence principles and applications in biology and medicine, A. K. Campbell, publ. 1988 Horwood/VCH Chichester Weinheim, for further discussion of bioluminescent reactions.
It has now been found that the light emitted from a bioluminescent reaction involving a modified bioluminescent or xe2x80x9crainbowxe2x80x9d protein, may be changed in intensity, colour or polarisation. Such a change can then be used in various assays for detecting, locating and measuring cells, microbes and biological molecules.
In this instance, the cell or substance causes a physical or chemical change, such as phosphorylation, to a rainbow protein such as a genetically engineered luciferase, resulting in a change in intensity, colour or polarisation of the light emission. The bioluminescent reaction is triggered by adding, for example, the luciferin, and modification of the luciferase by the cell or substance being measured causes the reaction to emit light at a shorter or longer wavelength. This enables specific reactions to be detected and quantified in live cells, and within organelles or on the inner or outer surface of the plasma membrane, without the need to break them open, and without the need for separation of bound and unbound fractions.
According to one aspect of the invention there is provided a bioluminescent protein capable of taking part in a bioluminescent reaction to produce light or radiation of altered characteristics under different physical, chemical, biochemical or biological conditions.
The rainbow protein may be produced by the alteration, substitution, addition or removal of one or more amino acids from the end of or within the luciferase or photoprotein. As a result the light emission from the oxyluciferin may be of different colours or different states of polarisation depending on the physical or chemical conditions. A change in colour to another part of the rainbow spectrum may be induced by:
(a) A change in cation concentration such as H, Ca Mg; or transition metal.
(b) A change in anion concentration such as C1xe2x88x92 or phosphate.
(c) Covalent modification of the new protein by enzymes causing phospho- or dephosphorylation (including ser/thr, his, and tyr kinases and phosphatases) transglutamination, proteolysis, ADP ribosylation, gly-or glu-cosylation, halogenation, oxidation, methylation and myristilation.
(d) Binding to the rainbow protein of an antigen, an intracellular signal such as cyclic AMP, cyclic GMP, Ip3, Ip4, diacyl glycerol, ATP, ADP, AMP, GTP, any oxy or deoxyribonucloside or nucleotide, a substrate, a drug, a nucleic acid, a gene regulator protein.
(e) Expression of its nucleic acid inside a live cell, as well as its modification or regulation within the cell by gene expression such as promoters, enhancers or oncogenes.
Single or multiple mutations and deletions may be detected in a piece of DNA (eg a PCR product) by linking the xe2x80x9crainbow proteinxe2x80x9d to one end of the DNA and an energy transfer acceptor or quencher to the other end. Muclease attack at the mutation will separate the rainbow protein from the acceptor or quencher and thus cause a change in intensity, colour or polarisation of the light emission.
Such alteration, substitution, addition or removal of one or more amino acids may be achieved by chemical means. Alteration of an acid includes alkylation (eg. methylation), phosphorylation and various other covalent modifications of the type outlined herein. Alternatively the nucleic acid coding for the luciferase or photoprotein may be altered by modifying, substituting, inserting or deleting one or more nucleotides such that the resulting protein has gained or lost a site which interacts with the cations, anions, intracellular signal, covalent modification; proteins or nucleic acid to be measured. The insertion or deletion of nucleotides is normally produced by site directed mutagenesis or by opening up the gene with a specific restriction enzyme, inserting or deleting a selected nucleotide sequence and then sealing up of the gene again or using specific primers with the polymerase chain reaction. The nucleic acid is then transcribed to mRNA and this is then translated to form the rainbow protein either inside a bacterial or eukaryotic cell, or in vitro using, for example, rabbit reticulocyte lysate. The new nucleic acid may contain an RNA polymerase promoter such as T7, SP6, or mammalian promotors such as actin, myosin, myelin proteins, MMT-V SV40, antibody G6P dehydrogenase, and can be amplified in vitro using the polymerase chain reaction. The result is that the rainbow protein can be produced either in a live cell such as a cancer cell, or without cells using enzymatic reactions in vitro. The addition of tissue specific promoter or enhancer sequences to the 5xe2x80x2 or 3xe2x80x2 end of the DNA coding for the native or altered bioluminescent protein will enable it to be used as a reporter gene and to be expressed specifically in a particular cell or tissue, the expression being detectable by the appearance of a change in light intensity, colour or polarisation.
Another way of producing the DNA for a rainbow protein is to separate into two halves the original DNA for the bioluminescent protein. A piece of DNA or gene is then inserted between the two halves by ligating one half to the 5xe2x80x2 end and the other to the 3xe2x80x2 end. Alternatively the rainbow protein DNA could be generated using the polymerase chain reaction so that the sense primer had one part of the rainbow protein DNA linked at 5xe2x80x2 end and the antisense primer and the other part linked at the 3xe2x80x2 end (i.e. antisense). The pieces of DNA or gene of interest, in the middle, could be from two separate genes. For example one could code for an energy transfer protein, the other for a bioluminescent protein. Only when the two are linked together via a peptide (from DNA/RNA) will the rainbow protein be generated and a shift in colour occur. The energy transfer protein could be any fluor bound covalently or non-covalently to the protein, for example the green fluorescent protein from Aequorea, Obelia, Penilla or other cnidarians, or the blue or yellow fluorescent protein from luminous bacteria, or a flavoprotein, or a phyobiliprotein. The whole protein or just the fluorescent demain may be used. The bioluminescent protein would be any luciferase for example bacterial, firefly, glowworm or copepod, or any photoprotein for example aequorin, obelin or a radiolarin such as thalassicollin.
The protein or its DNA or RNA may be incorporated into a live bacterium or eukaryotic cell by using a virus, plasmid, calcium phosphate transfection, electroporation, liposome fusion or membrane pore forming proteins. Once inside, only live cells with the appropriate biochemistry will produce the xe2x80x9crainbow effectxe2x80x9d. By incorporating the xe2x80x9crainbow proteinxe2x80x9d gene into an embryo, oocyte, sperm, seed or seedling a transgenic animal or plant may be produced, enabling gene expression, cell regulation, drug action, or cell damage to be located and measured in individual organs using the xe2x80x9crainbow effectxe2x80x9d. These new organisms may also be used in home aquaria, on aeroplane runways, as safe lights at sea, and as house plants.
The rainbow protein may also be incorporated in a different part of the cell by chemical means or genetically engineering the protein to contain a signal peptide which locates it to the inner or outer surface of the plasma membrane or within a particular intracellular organelle (e.g. peroxisome, mitochondrion, chloroplast, endoplasmic reticulum, golgi, secretory vesicle, nucleus or endosme.
Addition of a signal peptide, either chemically or by genetic engineering, will enable the normal or altered luciferase or photoprotein to be targeted into a specific organelle within the cell, or onto the inner or outer surface of the plasma membrane. For example the sequence MLSRLSLRLLSRYLL (SEQ ID NO: 1) at the N terminus will locate the bioluminescent protein in the mitochondria, and KKSALLALMYVCPGKADKE (SEQ ID NO: 2) on the N terminus will target the protein to the endoplasmic reticulum, a KDEL (SEQ ID NO: 3) sequence at the C terminus retaining it there.
The initial luciferase or photoprotein or its gene may come from any of the known chemistries in bioluminescence (see FIG. 1) or from the wide range of uncharacterised luminous organisms from more than 700 genera representing at least 16 phyla. The luciferin may be synthesised chemically and added to the biological reaction or cell to generate light. Aternatively, the gene coding for the enzymes responsible for making the luciferin may be linked to the xe2x80x9crainbow proteinxe2x80x9d gene so that the artificial operon or fusion gene expresses both rainbow protein and makes luciferin in the live cell from normal cell constituents such as amino acids.
According to a second aspect of the invention there is provided a method of producing a bioluminescent protein by altering, substituting, adding or deleting one or more amino acids to the protein by chemical means or by genetically engineering the nucleic acid coding for the protein.
According to a further aspect of the invention there is provided nucleic acid coding for the bioluminescent protein as hereinbefore defined.
The rainbow protein, or the nucleic acid coding for it, may be used in a range of biological investigations; such as:
(a) Detection, location and measurement of microbes (protozoa, bacteria, viruses).
(b) Detection and location of cancer cells.
(c) Measurement of enzymes, intracellular signalling and other turnover reactions in cells or fluids.
(d) DNA and RNA binding assays.
(e) Immunoassay and other protein binding assays.
The rainbow proteins and their parent nucleic acids also may be used in genetic engineering, in the development of transgenic animals, plants and microbes, and in horticulture.
According to yet a further aspect of the invention there is provided the use of a rainbow protein, or the nucleic acid coding for the rainbow protein, for the detection, location or measurement of substances of biological interest such as microbes, cells or biological molecules or reactions therein.
In this aspect, the reaction or substances of biological interest are made to interact with the rainbow protein or its parent nucleic acid. Such interactions include direct or indirect linking such as non-covalent or covalent binding as well as energy transfer processes.
Although the invention has been described above it is to be understood that it includes any inventive combination of the features set out above or in the following description.
The invention may be performed in various ways and will be further described with reference to the following examples: